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Isolation And Identification Of The Dominant Spoilage Biogenic Amine-producing Bacteria In Refrigerated Skipjack Tuna And Its Inhibition Study

Posted on:2023-12-23Degree:MasterType:Thesis
Country:ChinaCandidate:Y F LiuFull Text:PDF
GTID:2531306818493944Subject:Food Engineering
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Skipjack tuna is an important economic fish which is a kind of tuna.In this study,we firstly established a method for rapid detection of biogenic amines in skipjack tuna by ultrahigh performance liquid chromatography-tandem mass spectrometry(UPLC-MS).Then the dominant spoilage amine-producing bacteria were isolated and screened from skipjack tuna.After compared their spoilage and amine-producing ability,we selected the strain with the highest spoilage amine-producing for the study of the inhibition mechanism to provide theoretical basis for refrigerated skipjack tuna preservation.The specific research content and results were as follows:1.Firstly,an ultrahigh performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method was developed for the detection of eight biogenic amines in skipjack tuna.There were spermidine(SPM),spermidine(SPD),tyramine(TYR),tryptamine(TRP),histamine(HIS),β-phenylethylamine(PEA),putrescine(PUT),and cadaverine(CAD).The samples were extracted with 0.2%acidified acetonitrile and dichloromethane.The extracts were separated on a C18 column with gradient elution using methanol and 0.5%formic acid aqueous solution as mobile phases.The mass spectrometry data were obtained in multiple reaction monitoring(MRM)mode,and the samples were quantified by internal standard method.The results showed that the eight biogenic amines could be separated within 6 min with good linearity in concentration ranges,respectively.The correlation coefficients R were0.9956~0.9996.The limits of detection were 75~150μg/kg,and the limits of quantification were 250~500μg/kg.The average recoveries of the samples were82%~113%,and the relative standard deviations were 2.3%~9.7%.Based on the experiments of method validation,it can be concluded that this method has good sensitivity,recovery and precision.It can be used for the rapid determination of biogenic amines in skipjack tuna,providing a rapid detection method for subsequent experiments.2.Three strains of Pseudomonas fluorescens,Citrobacter freundii and Aeromonas hydrophila were selected from skipjack tuna and stored at low temperature.The strains were identified by 16S r DNA molecular identification technique.The dominant spoilage amine-producing bacteria were inoculated into sterile fish meat and stored at 4℃.The spoilage capacity of the three dominant spoilage amine-producing bacteria was analyzed by measuring the total number of colonies and volatile salt nitrogen(TVB-N),and the spoilage metabolite yield factor(YTVB-N/CFU).The amine production capacity of the three dominant spoilage amine-producing bacteria was compared by measuring the biogenic amine content in the samples.The results showed that at the end of storage,the total number of colonies of P.fluorescens,C.fumigatus and A.hydrophila reached8.36,8.27 and 8.13 log CFU/g,respectively.The TVB-N values were 28.21,30.30 and31.29 mg/100 g,respectively.The three dominant spoilage bacteria were A.hydrophila>C.fumigatus>P.fluorescens.On the 8th day of storage,the histamine contents of the P.fluorescens,C.freundii and A.hydrophila groups reached 196.23,83.43 and 261.22 mg/kg,respectively.A.hydrophila group’s total biogenic amine content at the end of storage was also greater than that of the groups inoculated with P.fluorescens and C.fumigatus.Therefore,A.hydrophila was selected as the main spoilage biogenic amine-producing producing bacteria in refrigerated skipjack tuna for the bacterial inhibition study.3.A Lactobacillus plantarum was screened from yogurt,and the cell-free supernatant(CFS)of L.plantarum was used as an inhibitor to investigate the inhibition mechanism of A.hydrophila.The minimum inhibitory concentration(MIC)was 10%CFS by 96-well plate test.The integrity of the cell membrane and cell morphology of the bacteria was observed by fluorescence electron microscopy and scanning electron microscopy,which found the cell membrane of the control group was intact and the cell morphology was rod shape.The cell membrane of the MIC and 2MIC groups were more severely damaged and the cell morphology of the bacteria was deformed and broken to different degrees.The results suggested that the basal peak chromatograms of the control and MIC groups were significantly different.The results of the orthogonal-partial least squares discriminant analysis showed that the data were feasible.The metabolomics experiment screened a total of 114 differential metabolites from the two groups,including 46 up-regulated differential metabolites and 68 down-regulated differential metabolites.The metabolic pathways of the differential metabolites using KEGG showed that amino acid metabolism,sugar metabolism and nucleic acid metabolism were affected in the MIC group of A.hydrophila.The activities of succinate dehydrogenase and malate dehydrogenase,which are important in the tricarboxylic acid cycle(TCA cycle),were reduced.It was indicated that CFS disrupted the cell membrane of the bacteria and affected the stability of metabolism in the bacteria,thus achieving the inhibition effect.Finally,CFS and A.hydrophila were reconstituted into sterile fish blocks to investigate the bacterial inhibitory effect of the inhibitor in fish meat.The results of the experimental group stored at 4℃for 12 days showed that CFS had a significant bacterial inhibitory effect on the 6 days.The total number of A.hydrophila colonies in fish meat started to increase gradually from day 6.The inhibition effect of CFS was most obvious on the 3 days.It was demonstrated that CFS of L.plantarum had significant antibacterial activity in both the bacterial solution and fish.
Keywords/Search Tags:skipjack tuna, biogenic amines, UPLC-MS/MS, dominant spoilage bacteria, biogenic amine-producing bacteria, Aeromonas hydrophila, Lactobacillus plantarum, bacterial inhibition mechanism
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